The human heart beats about three billion times in a normal human lifetime. Since this rhythm is myogenic in origin, all the proteins (ion channels and transporters) responsible for this rhythm reside in the myocyte membrane. It is the purpose of this proposal to investigate tile molecular and biophysical properties of one major component of cardiac pacemaker activity, the hyperpolarization-activated current I-f. The project aims to pursue a number of findings made in the laboratory over the recent past. We have demonstrated that: (1) I-f exists in all cardiac cell types, but it activates at more negative voltages as one proceeds distally in the conduction pathway; (2) that the multigene family that underlies the alpha subunit of I-f is differentially expressed in different cardiac regions; and (3) that the auxiliary subunit MiRP1 is highly expressed in sinus node, and can be demonstrated to form a complex with HCN family members when heterologously coexpressed in Xenopus oocytes. MiRP1 also enhances expression of the alpha subunit in this system. In the first three aims of the proposal we investigate the consequences of these observations. Using a combination of patch clamping, molecular biology and protein chemistry we aim to determine: (1) the importance of MiRP 1 to the functional properties of I-f in sinus node and other cardiac regions; (2) how co-expression of HCN ion channel subunits with MiRP 1 enhances expression in heterologous expression systems (increase in channel number, conductance or open probability); and (3) the cell specific factor that determines the positive voltage dependence of I-f in sinus node. In the fourth and final aim of the proposal we expand our comparative approach to mammals of widely different heart rates (canine, rabbit, and mouse) and ask what role the molecular and biophysical properties of I-f play in determining these heart rate differences. Our preliminary results with canine and rabbit sinus nodes suggest that I-f current density may be important. These studies should add significantly to our understanding of the molecular and biophysical origin of the heartbeat, and might point the way to new therapeutics for disturbances of cardiac rhythm.